Journal: Virologica Sinica

Article Title: Pseudorabies virus VHS protein abrogates interferon responses by blocking NF-κB and IRF3 nuclear translocation.

doi: 10.1016/j.virs.2024.05.009

Figure Lengend Snippet: Fig. 1. UL41 suppresses the mRNA levels of IFN-β, TNF-α, IL-6, IL-8 and ISGs. A PK-15 cells were transfected with pCMV-Myc empty vector or Myc tagged UL41 expression plasmid for 24 h. Then cells were either uninfected or infected with PRV (MOI ¼ 0.001) for 18 h and total RNA was extracted for RT-qPCR of the indicated genes. B, C Myc-tagged UL41 (200 ng, 500 ng, 800 ng and 1 μg) plasmid was transfected into PK-15 cells for 24 h, and then either left untreated or treated with poly(dA:dT) (B) or 2030-cGAMP (C) (2 mg/mL) for 12 h. The mRNA expression levels of indicated genes were determined by RT-qPCR. The expression of UL41 was detected with Myc specific antibody. GAPDH serves as a loading control. Data from at least three independent experiments are presented as the mean standard deviation. *P < 0.05, **P < 0.01, ***P < 0.001; ns, not significant. Western blot images are the representatives of three independent experiments.

Article Snippet: NF-κB p65 rabbit polyclonal antibody (1:1000, 10745-1-AP), GAPDH mouse monoclonal antibody (1:2000, 60004-1-Ig), Myc tag mouse monoclonal antibody (1:1000, 60003-2-Ig), NF-κB1 rabbit polyclonal antibody (1:1000, 14220-1-AP), IκB Alpha Polyclonal antibody (1:500, 10268-1-AP), TAK1 rabbit polyclonal antibody (1:1000, 12330-2-AP), Anti-Flag tag rabbit polyclonal antibody (1:1000, 80010-1-RR), Anti-Flag tag Mouse Monoclonal antibody (1:1000, 66008-4-Ig), and VDAC1/Porin polyclonal antibody (1:1000, 55259-1-AP) were purchased from Proteintech (Wuhan, China).

Techniques: Transfection, Plasmid Preparation, Expressing, Infection, Quantitative RT-PCR, Control, Standard Deviation, Western Blot

Journal: Virologica Sinica

Article Title: Pseudorabies virus VHS protein abrogates interferon responses by blocking NF-κB and IRF3 nuclear translocation.

doi: 10.1016/j.virs.2024.05.009

Figure Lengend Snippet: Fig. 2. UL41 degrades IRF3 and inhibites the cGAS-STING-mediated phosphorylation of IRF3. A PK-15 cells were cultured overnight in 6-well plates. The indicated plasmids (HA-cGAS, HA-STING, Flag-TBK1, and Flag-IRF3(5D) (500 ng) were co-transfected into PK-15 cells with Myc-tagged UL41 (1 μg) for 24 h. The expression of cGAS, STING, TBK1, IRF3, and UL41 proteins were detected using antibodies against HA, Myc and Flag. GAPDH serves as a loading control. The transcription level of IFN-β was detected by RT-qPCR. B, C Myc-tagged UL41 plasmid was transfected into PK-15 cells for 24 h, and then either left untreated or treated with poly(dA:dT) (2 mg/mL) (B) or 2030-cGAMP (2 mg/mL) (C) for 12 h. The expression of cGAS, STING, TBK1, IRF3, phosphorylation IRF3 and UL41 proteins were detected using indicated antibodies. GAPDH serves as a loading control. D, E HEK-293 cells were co-transfected with Flag-tagged IRF3 (5D) (1 μg) and Myc-tagged UL41 (1 μg) expression plasmid for 24 h. Cells were processed for IP with anti-Flag or anti-Myc beads. Inputs and precipitated proteins were probed with antibodies against Flag- tag, Myc-tag, and GAPDH. F Myc-tagged UL41 (1 μg) plasmid was transfected into PK-15 cells for 24 h and either left untreated or treated with 2030-cGAMP (2 mg/mL). Cells were stained with anti-Myc (red), anti-IRF3 (green) and anti-p-IRF3 (green) subjected to analysis by confocal microscopy. Nuclei were stained with DAPI (blue). Scale bars, 10 μm. G Myc-tagged UL41 plasmid (1 μg) was transfected into PK-15 cells for 24 h, and then either left untreated or treated with 2030-cGAMP (2 g/mL). Then, cytoplasmic and nuclear proteins were extracted and subjected to Western blot analysis. Expression of IRF3 and Myc-tagged UL41 was detected with specific antibodies. Histone 3 was used as a nuclear protein marker. HSP90β served as a cytoplasm marker. þ represents for transfected, and – represents for untransfected. Data from at least three independent experiments are presented as the mean standard deviation. ***P < 0.001. Western blot, fluorescence images, and Co-IP results are representatives of three independent experiments.

Article Snippet: NF-κB p65 rabbit polyclonal antibody (1:1000, 10745-1-AP), GAPDH mouse monoclonal antibody (1:2000, 60004-1-Ig), Myc tag mouse monoclonal antibody (1:1000, 60003-2-Ig), NF-κB1 rabbit polyclonal antibody (1:1000, 14220-1-AP), IκB Alpha Polyclonal antibody (1:500, 10268-1-AP), TAK1 rabbit polyclonal antibody (1:1000, 12330-2-AP), Anti-Flag tag rabbit polyclonal antibody (1:1000, 80010-1-RR), Anti-Flag tag Mouse Monoclonal antibody (1:1000, 66008-4-Ig), and VDAC1/Porin polyclonal antibody (1:1000, 55259-1-AP) were purchased from Proteintech (Wuhan, China).

Techniques: Phospho-proteomics, Cell Culture, Transfection, Expressing, Control, Quantitative RT-PCR, Plasmid Preparation, FLAG-tag, Staining, Confocal Microscopy, Western Blot, Marker, Standard Deviation, Co-Immunoprecipitation Assay

Journal: Virologica Sinica

Article Title: Pseudorabies virus VHS protein abrogates interferon responses by blocking NF-κB and IRF3 nuclear translocation.

doi: 10.1016/j.virs.2024.05.009

Figure Lengend Snippet: Fig. 3. UL41 recruits Tollip to promote IRF3 autophagic degradation. A PK-15 cells were co-transfected with Flag tagged IRF3(5D) (1 μg), Myc-tagged UL41 (1 μg) or pCMV-Myc empty vector (EV) (1 μg) for 24 h, then cells were treated with proteasomal inhibitor MG132 (7.5 μmol/L), lysosome inhibitor CQ (50 μmol/L) or caspase inhibitor Z-VAD-FMK (20 μmol/L) for 12 h. DMSO treated cells served as vehicle control. Cells were collected and immunoblotted for Flag-tagged IRF3 and Myc- tagged UL41. GAPDH served as loading control. B, C HEK-293 cells were co-transfected with Myc-tagged autophagy receptor expression plasmids (Tollip, NDP52, p62, OPTN and NBR1) (1 μg) and Flag-tagged UL41 (1 μg) expression plasmids for 24 h. Cells were processed for IP with anti-Flag or anti-Myc beads. Inputs and precipitated proteins were probed with antibodies against Flag-tag, Myc-tag, and GAPDH. D PK-15 cells were co-transfected with Tollip or siTollip, Flag-tagged IRF3(5D) and Myc-tagged UL41 for 24 h. LC3, IRF3, Tollip and Myc tagged UL41 were assessed by immunoblot analysis. GAPDH served as a loading control. E HEK-293 cells were co-transfected with Myc-tagged autophagy receptor expression plasmids (Tollip, NDP52, p62) (1 μg) or Flag-tagged IRF3(5D) (1 μg) and Myc- tagged UL41 (1 μg) for 24 h. IRF3, Tollip, NDP52, p62, Myc tagged UL41 and GAPDH were assessed by immunoblot analysis. F HEK-293 cells were transfected with siNC, siTollip, siNDP52, sip62 or Flag-tagged IRF3(5D) (1 μg) and Myc-tagged UL41 (1 μg) as indicated for 24 h. IRF3, Tollip, NDP52, p62, Myc tagged UL41 and GAPDH were assessed by immunoblot analysis. Western blot and Co-IP results are representatives of three independent experiments.

Article Snippet: NF-κB p65 rabbit polyclonal antibody (1:1000, 10745-1-AP), GAPDH mouse monoclonal antibody (1:2000, 60004-1-Ig), Myc tag mouse monoclonal antibody (1:1000, 60003-2-Ig), NF-κB1 rabbit polyclonal antibody (1:1000, 14220-1-AP), IκB Alpha Polyclonal antibody (1:500, 10268-1-AP), TAK1 rabbit polyclonal antibody (1:1000, 12330-2-AP), Anti-Flag tag rabbit polyclonal antibody (1:1000, 80010-1-RR), Anti-Flag tag Mouse Monoclonal antibody (1:1000, 66008-4-Ig), and VDAC1/Porin polyclonal antibody (1:1000, 55259-1-AP) were purchased from Proteintech (Wuhan, China).

Techniques: Transfection, Plasmid Preparation, Control, Expressing, FLAG-tag, Western Blot, Co-Immunoprecipitation Assay

Journal: Virologica Sinica

Article Title: Pseudorabies virus VHS protein abrogates interferon responses by blocking NF-κB and IRF3 nuclear translocation.

doi: 10.1016/j.virs.2024.05.009

Figure Lengend Snippet: Fig. 4. UL41 Dampens TNF-α-mediated NF-κB activation. A HEK-293 and PK-15 cells were transfected with pCMV-Myc empty vector (1 μg) or Myc tagged UL41 (1 μg) expression plasmid. At 24 h post-transfection, the cells were treated with TNF-α (10 ng/mL) for 12 h. Total cellular RNA was extracted and transcription level of IL-6 and IL-8 was analyzed by RT-qPCR. B HEK-293 and PK-15 cells were transfected with pCMV-Myc empty vector (1 μg) or Myc tagged UL41 (1 μg) expression plasmid. At 24 h post-transfection, the expression of MyD88, TAK1, IκBα, p-IκBα, p65, p50 and Myc tagged UL41 were detected by western blotting. GAPDH serves as a loading control. C HEK-293 and PK-15 cells were transfected with pCMV-Myc empty vector (1 μg) or Myc tagged UL41 (1 μg) expression plasmid. At 24 h post-transfection, the cells were treated with TNF-α (10 ng/mL) for 12 h. The expression of MyD88, TAK1, IκBα, p-IκBα, p65, p50 and Myc tagged UL41 were detected by Western blotting. GAPDH serves as a loading control. Data from at least three independent experiments are presented as the mean standard deviation. ***P < 0.001. Western blot results are representatives of three independent experiments.

Article Snippet: NF-κB p65 rabbit polyclonal antibody (1:1000, 10745-1-AP), GAPDH mouse monoclonal antibody (1:2000, 60004-1-Ig), Myc tag mouse monoclonal antibody (1:1000, 60003-2-Ig), NF-κB1 rabbit polyclonal antibody (1:1000, 14220-1-AP), IκB Alpha Polyclonal antibody (1:500, 10268-1-AP), TAK1 rabbit polyclonal antibody (1:1000, 12330-2-AP), Anti-Flag tag rabbit polyclonal antibody (1:1000, 80010-1-RR), Anti-Flag tag Mouse Monoclonal antibody (1:1000, 66008-4-Ig), and VDAC1/Porin polyclonal antibody (1:1000, 55259-1-AP) were purchased from Proteintech (Wuhan, China).

Techniques: Activation Assay, Transfection, Plasmid Preparation, Expressing, Quantitative RT-PCR, Western Blot, Control, Standard Deviation

Journal: Virologica Sinica

Article Title: Pseudorabies virus VHS protein abrogates interferon responses by blocking NF-κB and IRF3 nuclear translocation.

doi: 10.1016/j.virs.2024.05.009

Figure Lengend Snippet: Fig. 5. UL41 targets p50 to inhibit NF-κB signaling. A PK-15 cells were transfected with Myc tagged UL41 (1 μg) or pCMV-Myc empty vector (EV) (1 μg) for 12 h following by stimulating with TNF-α (10 ng/mL) for another 12 h. Cells were treated with proteasomal inhibitor MG132 (7.5 μmol/L), lysosome inhibitor CQ (50 μmol/L) or caspase inhibitor Z-VAD-FMK (50 μmol/L) for 12 h. DMSO treated cells served as vehicle control. Then, cells were collected and immunoblotted for p50 and Myc-tagged UL41. GAPDH served as loading control. B HEK-293 cells were transfected with Myc-tagged UL41 (1 μg) expression plasmid for 12 h following by stimulating with TNF-α (10 ng/mL) for another 12 h. Cells were treated with Z-VAD-FMK (50 μmol/L) for 12 h, and then processed for IP with anti-Myc or anti-p50 beads. Inputs and precipitated proteins were probed with antibodies against Myc-tag, p50, and GAPDH. C PK-15 cells were cotransfected with Myc-tagged UL41 (1 μg) expression plasmid for 24 h. Then, the cells were treated with TNF-α (10 ng/mL) for another 12 h. Cells were stained with anti-Myc (red) and anti-p50 (green) subjected to analysis by confocal microscopy. Nuclei were stained with DAPI (blue). Scale bars, 10 μm. Western blot, fluorescence images, and Co-IP results are representatives of three independent experiments.

Article Snippet: NF-κB p65 rabbit polyclonal antibody (1:1000, 10745-1-AP), GAPDH mouse monoclonal antibody (1:2000, 60004-1-Ig), Myc tag mouse monoclonal antibody (1:1000, 60003-2-Ig), NF-κB1 rabbit polyclonal antibody (1:1000, 14220-1-AP), IκB Alpha Polyclonal antibody (1:500, 10268-1-AP), TAK1 rabbit polyclonal antibody (1:1000, 12330-2-AP), Anti-Flag tag rabbit polyclonal antibody (1:1000, 80010-1-RR), Anti-Flag tag Mouse Monoclonal antibody (1:1000, 66008-4-Ig), and VDAC1/Porin polyclonal antibody (1:1000, 55259-1-AP) were purchased from Proteintech (Wuhan, China).

Techniques: Transfection, Plasmid Preparation, Control, Expressing, Staining, Confocal Microscopy, Western Blot, Co-Immunoprecipitation Assay

Journal: Virologica Sinica

Article Title: Pseudorabies virus VHS protein abrogates interferon responses by blocking NF-κB and IRF3 nuclear translocation.

doi: 10.1016/j.virs.2024.05.009

Figure Lengend Snippet: Fig. 6. UL41 promotes apoptosis in PK-15 cells. A PK-15 cells were infected with PRV (MOI ¼ 1). Flow cytometry was used to determine apoptotic events by annexin V (AV-FITC) and propidium iodide (PI-PE) staining in PK-15 cells induced by PRV at 0, 3, 9, 15 and 21 h after infection. B Flow cytometry was used to detect apoptosis of PK-15 induced by PRV at 0, 3, 9, 15 and 21 h after infection. C PK-15 cells were infected with PRV (MOI ¼ 1) for 0 h, 3 h, 8 h, 15 h and 21 h. The mRNA levels of PRV UL41 were assessed by RT-qPCR. D PK-15 cells were transfected with Myc tagged UL41 (1 μg) or pCMV-Myc empty vector (1 μg) for 24 h. Flow cytometry was used to determine apoptotic events by annexin V (AV-FITC) and propidium iodide (PI-PE) staining. E, F PK-15 cells were transfected with pCMV-Myc empty vector (1 μg) or Myc-tagged UL41 (1 μg) expression plasmid. At 24 h post-transfection, cells were uninfected or infected with PRV (MOI ¼ 1) for 9 h and total RNA was extracted and the mRNA levels of Bcl-2 and Cyto-C were assessed by RT-qPCR. G PK-15 cells were transfected with pCMV-Myc empty vector (1 μg) or Myc-tagged UL41 (1 μg) expression plasmid. At 24 h post-transfection, cells were uninfected or infected with PRV (MOI ¼ 1) for 9 h. The protein expression level of Bcl-2 was detected by Western blot. GAPDH serves as a loading control. H PK-15 cells were transfected with pCMV-Flag empty vector (1 μg) or Flag-tagged UL41 (1 μg) expression plasmid for 24 h. Then, cytoplasmic and mitochondria proteins were extracted and subjected to Western blot analysis. Expression of Cyto-C and Flag-tagged UL41 was detected with specific antibodies. VDAC1 was used as a mitochondria protein marker. HSP90β served as a cytoplasm marker. I PK-15 cells were transfected with Myc tagged UL41 (1 μg) or pCMV-Myc empty vector (EV) (1 μg) for 12 h following by stimulating with TNF-α (10 ng/mL) for another 12 h. Cells were treated with Caspase3 inhibitor Ac-DEVD-CHO (20 nmol/L), Caspase8 inhibitor Z-IETD-FMK (20 nmol/L) or caspase9 inhibitor Z-LEHD-FMK TFA (20 nmol/L) for 12 h. DMSO treated cells served as vehicle control. Then, cells were collected and immunoblotted for p50 and Myc-tagged UL41. GAPDH served as a loading control. Data from at least three independent experiments are presented as the mean standard deviation. *P < 0.05, ***P < 0.001; ns, not significant. Western blot results are representatives of three independent experiments.

Article Snippet: NF-κB p65 rabbit polyclonal antibody (1:1000, 10745-1-AP), GAPDH mouse monoclonal antibody (1:2000, 60004-1-Ig), Myc tag mouse monoclonal antibody (1:1000, 60003-2-Ig), NF-κB1 rabbit polyclonal antibody (1:1000, 14220-1-AP), IκB Alpha Polyclonal antibody (1:500, 10268-1-AP), TAK1 rabbit polyclonal antibody (1:1000, 12330-2-AP), Anti-Flag tag rabbit polyclonal antibody (1:1000, 80010-1-RR), Anti-Flag tag Mouse Monoclonal antibody (1:1000, 66008-4-Ig), and VDAC1/Porin polyclonal antibody (1:1000, 55259-1-AP) were purchased from Proteintech (Wuhan, China).

Techniques: Infection, Flow Cytometry, Staining, Quantitative RT-PCR, Transfection, Plasmid Preparation, Expressing, Western Blot, Control, Marker, Standard Deviation

Journal: Virologica Sinica

Article Title: Pseudorabies virus VHS protein abrogates interferon responses by blocking NF-κB and IRF3 nuclear translocation.

doi: 10.1016/j.virs.2024.05.009

Figure Lengend Snippet: Fig. 7. E192, D194, and D195 sites of UL41 are critical for natural immune evasion. A Comparison of the amino acid sequences of the PRV UL41 protein and its homologs in alpha herpesviruses. B, C PK-15 cells were transfected with Flag-tagged-UL41-M (500 ng, 1 μg) expression plasmid. At 24 h post transfection, the cells were treated with TNF-α (10 ng/mL) for 12 h, and total cellular RNA was extracted. The fold change in gene expression of IL-6 or IL-8 was determined by RT-qPCR. The protein expression level of UL41-M was detected with Flag tag antibody. GAPDH serves as a loading control. D PK-15 cells were transfected with pCMV-Myc empty vector (1 μg) or Flag-tagged-UL41 (1 μg) and Flag-tagged-UL41-Mutant (1 μg) expression plasmid. At 24 h post transfection, the cells were treated with TNF-α (10 ng/ mL) for 12 h. The expression of p50 was detected with specific antibody. UL41 and UL41-M expression were detected with Flag tag antibody. GAPDH serves as a loading control. E PK-15 cells were cultured overnight in 6-well plates. Flag-IRF3(5D) (1 μg) were co-transfected into cells with the indicated doses of Flag-tagged UL41 (1 μg) or Flag-tagged UL41-Mutant (1 μg) expression plasmid for 24 h. Cells were collected for IFN-β mRNA detection by RT-qPCR. IRF3, p-IRF3, Flag tag- ged UL41 and UL41-M were detected with indicated antibodies. GAPDH serves as a loading control. F HEK-293 cells were transfected with Flag-tagged UL41 (1 μg) or Flag-tagged UL41-M (1 μg) expression plasmids for 24 h. Cells were processed for IP with anti-IRF3 beads. Inputs and precipitated proteins were probed with an- tibodies against Flag-tag, IRF3, and GAPDH. G PK-15 cells were transfected with Flag-tagged UL41 (1 μg) or Flag-tagged UL41-M (1 μg) expression plasmids for 24 h. Cells were stained with anti-Flag (red) and anti-IRF3 (green) subjected to analysis by confocal microscopy. Nuclei were stained with DAPI (blue). Scale bars, 10 μm. H Exogenous expression of UL41 enhances PRV replication in vitro. PK-15 cells were transfected with 1 μg Flag tagged UL41 and Flag tagged UL41-M plasmid for 24 h. Then, cells were infected with PRV (MOI ¼ 1) for 3 h, 6 h, 9 h and 12 h. TCID50 assay was performed for viral titer detection. Data from at least three independent experiments are presented as the mean standard deviation. *P < 0.05, **P < 0.01, ***P < 0.001; ns, not significant. Western blot, fluorescence images, and Co-IP results are representatives of three independent experiments.

Article Snippet: NF-κB p65 rabbit polyclonal antibody (1:1000, 10745-1-AP), GAPDH mouse monoclonal antibody (1:2000, 60004-1-Ig), Myc tag mouse monoclonal antibody (1:1000, 60003-2-Ig), NF-κB1 rabbit polyclonal antibody (1:1000, 14220-1-AP), IκB Alpha Polyclonal antibody (1:500, 10268-1-AP), TAK1 rabbit polyclonal antibody (1:1000, 12330-2-AP), Anti-Flag tag rabbit polyclonal antibody (1:1000, 80010-1-RR), Anti-Flag tag Mouse Monoclonal antibody (1:1000, 66008-4-Ig), and VDAC1/Porin polyclonal antibody (1:1000, 55259-1-AP) were purchased from Proteintech (Wuhan, China).

Techniques: Comparison, Transfection, Expressing, Plasmid Preparation, Gene Expression, Quantitative RT-PCR, FLAG-tag, Control, Mutagenesis, Cell Culture, Staining, Confocal Microscopy, In Vitro, Infection, TCID50 Assay, Standard Deviation, Western Blot, Co-Immunoprecipitation Assay

Journal: Virologica Sinica

Article Title: Pseudorabies virus VHS protein abrogates interferon responses by blocking NF-κB and IRF3 nuclear translocation.

doi: 10.1016/j.virs.2024.05.009

Figure Lengend Snippet: Fig. 8. Knockdown of UL41 promotes IFN-I pathway and NF-κB signaling activation. A PK-15 Cells were cultured overnight in 6-well plates, and si-UL41 and si-NC (non-targeting control) were transfected for 12 h. Then the cells were stimulated without or with poly(I:C) for 12 h, and uninfected or infected with PRV for 12 h. Cells were collected and total RNA were extracted for IFN-β and IL-6 mRNA detection by RT-qPCR. B PK-15 Cells were cultured overnight in 6-well plates, and si-UL41 and si-NC (nontargeting control) were transfected for 24 h. Subsequently, cells were infected without or with PRV (MOI ¼ 1) for 6 h or 12 h. IRF3, p50 and p65 phosphorylation levels were examined using western blotting assay. GAPDH serves a loading control. Data from at least three independent experiments are presented as the mean standard deviation. *P < 0.05, ***P < 0.001; ns, not significant. Western blot results are representatives of three independent experiments.

Article Snippet: NF-κB p65 rabbit polyclonal antibody (1:1000, 10745-1-AP), GAPDH mouse monoclonal antibody (1:2000, 60004-1-Ig), Myc tag mouse monoclonal antibody (1:1000, 60003-2-Ig), NF-κB1 rabbit polyclonal antibody (1:1000, 14220-1-AP), IκB Alpha Polyclonal antibody (1:500, 10268-1-AP), TAK1 rabbit polyclonal antibody (1:1000, 12330-2-AP), Anti-Flag tag rabbit polyclonal antibody (1:1000, 80010-1-RR), Anti-Flag tag Mouse Monoclonal antibody (1:1000, 66008-4-Ig), and VDAC1/Porin polyclonal antibody (1:1000, 55259-1-AP) were purchased from Proteintech (Wuhan, China).

Techniques: Knockdown, Activation Assay, Cell Culture, Control, Transfection, Infection, Quantitative RT-PCR, Phospho-proteomics, Western Blot, Standard Deviation

Journal: Matrix biology : journal of the International Society for Matrix Biology

Article Title: SERPINA3 is a marker of cartilage differentiation and is essential for the expression of extracellular matrix genes during early chondrogenesis.

doi: 10.1016/j.matbio.2024.07.004

Figure Lengend Snippet: Fig. 1. SERPINA3 (alpha-1 antichymotrypsin) is induced during chondrogenic differentiation. (A) Interrogation of a previously published RNAseq dataset of transcriptome changes of human MSCs undergoing chondrogenic differentiation (GSE109503; [18]), demonstrated that of the serpins expressed in cartilage, SER- PINA3 was particularly highly induced during chondrogenesis, as early as Day 1. (B) Induction of SERPINA3 was confirmed in independent experiments by qPCR. (C) Immunohistochemistry of Day 14 cartilage pellets revealed strong cellular staining for SERPINA3 with little staining evident in the extracellular matrix. Sections were also stained with an aggrecan antibody for comparison. D) Indeed, intracellular SERPINA3 staining was confirmed in adult human articular chondrocytes by spinning disk confocal microscopy using an antibody raised against SERPINA3 (i, ii). Hoechst was used to identify the nuclei, serving as a mask for downstream quantification of nuclear SERPINA3 (iii). Fluorescence channels were split to highlight SERPINA3 and Hoechst in the magnified regions of panels i and ii (top right) whilst the bottom right panel shows three cells with nuclear staining of SERPINA3 (from panel ii) with their corresponding fluorescence in the z-direction (iv). Data are representative of 2–3 independent experiments.

Article Snippet: Sections were blocked with horse serum for 20 min, followed by a rabbit SERPINA3 antibody (Proteintech 12,192–1-AP; 1:2000) for overnight at 4 ◦C.

Techniques: Immunohistochemistry, Staining, Comparison, Confocal Microscopy, Fluorescence

Journal: Matrix biology : journal of the International Society for Matrix Biology

Article Title: SERPINA3 is a marker of cartilage differentiation and is essential for the expression of extracellular matrix genes during early chondrogenesis.

doi: 10.1016/j.matbio.2024.07.004

Figure Lengend Snippet: Fig. 4. Bulk RNA sequencing demonstrates a role for SERPINA3 in driving chondrogenic gene expression changes. A) Workflow overview for RNA sequencing experiments. MSCs were transfected with siSERPINA3 or siControl siRNA for 48 h prior to the induction of chondrogenesis. Samples were collected at Day 0 (no chondrogenesis, MSCs), Day 3 and Day 7 of pellet culture, RNA extracted and sequenced from 3 independent donors. (B) SERPINA3 knockdown led to sig- nificant changes in gene expression at day 3 or day 7 of chondrogenesis, as shown by volcano plots. C) Changes in selected chondrogenic genes (COL2A1, ACAN, MATN3, COL9A1, COL11A1, SOX5, SOX6, SOX9) are shown as heatmaps, with adjusted p values following DESeq2 analysis shown (Day 7; siSERPINA3 vs siControl). D) Reactome pathway analysis supports a role for SERPINA3 in extracellular matrix regulation (downregulated pathways at day 7 shown with normalized enrichment score (NES)).

Article Snippet: Sections were blocked with horse serum for 20 min, followed by a rabbit SERPINA3 antibody (Proteintech 12,192–1-AP; 1:2000) for overnight at 4 ◦C.

Techniques: RNA Sequencing, Gene Expression, Transfection, Knockdown

Journal: Matrix biology : journal of the International Society for Matrix Biology

Article Title: SERPINA3 is a marker of cartilage differentiation and is essential for the expression of extracellular matrix genes during early chondrogenesis.

doi: 10.1016/j.matbio.2024.07.004

Figure Lengend Snippet: Fig. 5. SOX9 induction is abrogated following silencing of SERPINA3 at early time points in chondrogenesis. A) Protein was extracted from MSCs (day 0) or chondrogenesis pellets at day 1 and day 2. Silencing of SERPINA3 led to abrogation of SOX9 induction. Phosphorylation of Akt was also reduced following SERPINA3 silencing. Results representative of 3 independent experiments. B) Densitometric analysis of the blots in (A) with each donor normalised to siControl at each timepoint.

Article Snippet: Sections were blocked with horse serum for 20 min, followed by a rabbit SERPINA3 antibody (Proteintech 12,192–1-AP; 1:2000) for overnight at 4 ◦C.

Techniques: Phospho-proteomics

Journal: Advanced Science

Article Title: Deciphering the Role and Mechanism of Decidual Monocyte‐Derived Macrophage Infiltration in Obstetric Antiphospholipid Syndrome at Single‐Cell Resolution

doi: 10.1002/advs.202503480

Figure Lengend Snippet: Abnormal infiltration of monocyte‐derived CCR2 + macrophages in OAPS decidua and their pro‐inflammatory roles. A) Bar plot showing the proportions of MDMs in decidual immune cells from OAPS patients and HCs in scRNA‐seq data. B) FCM analysis of the proportion of CCR2 + macrophages in the decidua from OAPS patients (n = 11) compared with HCs (n = 18). C) Representative images for CCR2 + and CCR2 − macrophages from the decidua of OAPS patients and HCs under the transmission electron microscope. Scale bar: 2 µm. D) Bubble plot showing the functional gene expressions in different types of decidual myeloid cells based on scRNA‐seq data. E) Bar plot showing the GO terms of marker genes in decidual MDMs. F) Bar plot showing the KEGG terms of marker genes in decidual MDMs. G) GSEA indicates the term of inflammatory response is significantly enriched in decidual CCR2 + macrophages. H) Bar plot showing the relative expression levels of genes related to inflammatory factors in decidual CCR2 + and CCR2 − macrophages, as detected using RT‐qPCR (n = 3). I) Representative immunoblots and semi‐quantified results of Arginase 1, NF‐κB P65, and STAT3 expressions in decidual CCR2 + and CCR2 − macrophages from OAPS patients (n = 3). J) Representative immunofluorescence images for CD14 (green), CCR2 (red), IGFBP1 (croci), HLA‐G (cyan), and DAPI (blue) co‐staining in decidua from HCs. Scale bar: 1000 µm. K) Representative immunofluorescence images for CD14 (green), CCR2 (red), IGFBP1 (croci), HLA‐G (cyan), and DAPI (blue) co‐staining in decidua from OAPS patients. Scale bar: 2000 µm. L) Line chart showing the effects of medium supernatants from CCR2 + and CCR2 − macrophages on HTR‐8/SVneo proliferation, as measured by CCK‐8 (n = 3). M) Bar plot showing relative expression levels of CASP3 , MMP2 , and MMP9 genes in HTR‐8/SVneo treated with supernatants from CCR2 + and CCR2 − macrophages, as detected by RT‐qPCR (n = 3). Data in (A) and (B) are presented as mean ± SD and analyzed using Student's t ‐test. Data in (H) and (I) are presented as mean ± SD and analyzed using a paired t ‐test. Data in (L) and (M) are presented as mean ± SD and analyzed using one‐way ANOVA with Dunnett's multiple comparisons test. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001. OAPS, obstetric antiphospholipid syndrome; HCs, healthy controls; MDM, monocyte‐derived macrophage; FCM, flow cytometry; scRNA‐seq, single‐cell RNA sequencing; GO, Gene Ontology; KEGG, Kyoto Encyclopedia of Genes and Genomes; GSEA, Gene Set Enrichment Analysis; RT‐qPCR, real‐time quantitative polymerase chain reaction; SD, standard deviation; ANOVA, analysis of variance.

Article Snippet: CD14 Mouse Monoclonal antibody (Proteintech, China, #60253‐1‐Ig, 1:500), IGFBP1 Rabbit Polyclonal Antibody (Proteintech, China, #13981‐1‐AP, 1:2000), CCR2b‐specific Polyclonal antibody (Proteintech, China, #16154‐1‐AP, 1:800), HLA‐G Mouse Monoclonal antibody (Proteintech, China, #66447‐1‐Ig, 1:800), MCP‐1 Rabbit Polyclonal antibody (Proteintech, China, #26161‐1‐AP, 1:500), vWF Rabbit Polyclonal Antibody (Proteintech, China, #27186‐1‐AP, 1:300), DARC Polyclonal antibody (Proteintech, China, #55185‐1‐AP, 1:200), F4/80 Polyclonal antibody (CST, USA, #70 076, 1:600) and CCR2a‐specific Polyclonal antibody (Proteintech, China, #16153‐1‐AP, 1:800) were used as primary antibodies.

Techniques: Derivative Assay, Transmission Assay, Microscopy, Functional Assay, Marker, Expressing, Quantitative RT-PCR, Western Blot, Immunofluorescence, Staining, CCK-8 Assay, Flow Cytometry, RNA Sequencing, Real-time Polymerase Chain Reaction, Standard Deviation

Journal: Biomedicine & pharmacotherapy = Biomedecine & pharmacotherapie

Article Title: Maduramicin-guided nanotherapy: A polymeric micelles for targeted drug delivery in canine mammary tumors.

doi: 10.1016/j.biopha.2023.116062

Figure Lengend Snippet: Fig. 2. Characterization of MAD loaded TPGS polymeric micelles (MAD-TPGS). (A) The hydrodynamic diameter and (B) zeta potential of MAD-TPGS were deter mined by dynamic light scattering (DLS). (C) The morphology of MAD-TPGS was observed by transmission electron microscopy (TEM). Scale bar = 100 nm. (D) The size distribution histogram was obtained via size analysis of TEM images of the particles. (E) X-ray diffraction (XRD) spectra and (F) Fourier transform infrared (FT- IR) spectra of MAD-TPGS, free TPGS polymer micelles, MAD and a physical mixture of MAD and free TPGS polymer micelles.

Article Snippet: MAD, TPGS, the physical mixture of MAD and TPGS, sucrose and lyophilized MAD-TPGS were measured via XRD (D8 Advance, Bruker-AXS, Karlsruhe, Germany) X.

Techniques: Zeta Potential Analyzer, Transmission Assay, Electron Microscopy, Fourier Transform Infrared Spectroscopy, Polymer

Journal: Biomedicine & pharmacotherapy = Biomedecine & pharmacotherapie

Article Title: Maduramicin-guided nanotherapy: A polymeric micelles for targeted drug delivery in canine mammary tumors.

doi: 10.1016/j.biopha.2023.116062

Figure Lengend Snippet: Fig. 4. Suppressed growth and restrained migration and invasion of CMT-U27 cells by MAD-TPGS. (A) The viability of CMT-U27 cells was determined with TPGS, MAD and MAD-TPGS at the indicated concentrations. Data were expressed as the mean (n = 6) ± standard deviation (SD). (B) The proliferation of CMT-U27 cells treated with TPGS, MAD (1 μM) or MAD-TPGS (1 μM) was determined by a colony formation assay. (C) Typical images of the migration and (D) invasion of CMT-U27 cells were taken by microscopy. Scale bar = 200 µm. (E) Photomicrograph of the wound-healing scratch healing assay of CMT-U27 cells was treated with TPGS, MAD (1 μM), or MAD-TPGS (1 μM). Scale bar = 100 µm. All representative images were selected from three independent experiments.

Article Snippet: MAD, TPGS, the physical mixture of MAD and TPGS, sucrose and lyophilized MAD-TPGS were measured via XRD (D8 Advance, Bruker-AXS, Karlsruhe, Germany) X.

Techniques: Migration, Standard Deviation, Colony Assay, Microscopy

Journal: Biomedicine & pharmacotherapy = Biomedecine & pharmacotherapie

Article Title: Maduramicin-guided nanotherapy: A polymeric micelles for targeted drug delivery in canine mammary tumors.

doi: 10.1016/j.biopha.2023.116062

Figure Lengend Snippet: Fig. 5. Oxidative stress Induced by MAD-TPGS in CMT-U27 cells. (A) Reactive oxygen species (ROS) and (B) the mitochondrial membrane potential (MMP) of CMT- U27 cells treated with TPGS, MAD (1 μM) or MAD-TPGS (1 μM) for 24 h were detected by immunofluorescence technique. Scale bar = 200 µm. All representative images were selected from three independent experiments. (C) Apoptosis assay was performed in CMT-U27 cells treated with TPGS, MAD (1 μM) and MAD-TPGS (1 μM). (D) The graph represents the percentage of Annexin V positive cells. Data were expressed as the mean (n = 3) ± standard deviation (SD). * P < 0.05, * * P < 0.01 vs control, ## P < 0.01 for MAD-TPGS vs free MAD.

Article Snippet: MAD, TPGS, the physical mixture of MAD and TPGS, sucrose and lyophilized MAD-TPGS were measured via XRD (D8 Advance, Bruker-AXS, Karlsruhe, Germany) X.

Techniques: Membrane, Immunofluorescence, Apoptosis Assay, Standard Deviation, Control

Journal: Biomedicine & pharmacotherapy = Biomedecine & pharmacotherapie

Article Title: Maduramicin-guided nanotherapy: A polymeric micelles for targeted drug delivery in canine mammary tumors.

doi: 10.1016/j.biopha.2023.116062

Figure Lengend Snippet: Fig. 6. Efficacy and antimetastatic effect of MAD-TPGS on nude BALB/c mice bearing orthotopic tumors of CMT-U27 cells. (A) Illustration of saline, TPGS, MAD (1.75 mg/kg) or MAD-TPGS (1.75 mg/kg) for oral administration or (F) tail vein injection of CMT-U27 tumor bearing nude mice (n = 5 mice per group). (B) Tumor volume curves of all groups over time after oral administration or (G) tail vein injection (n = 5). (C) Photographs of tumors were taken from different groups treated with oral administration or (H) tail vein injection (n = 5). (D) Tumor weights of the different groups via oral administration or (I) tail vein injection on Day 18 (n = 5), * P < 0.05 for MAD-TPGS vs MAD. (E) H&E, E-cadherin, Vimentin, and N-cadherin staining was performed on tumor sections after oral and tail vein treatment. Scale bar = 100 µm.

Article Snippet: MAD, TPGS, the physical mixture of MAD and TPGS, sucrose and lyophilized MAD-TPGS were measured via XRD (D8 Advance, Bruker-AXS, Karlsruhe, Germany) X.

Techniques: Saline, Injection, Staining

Journal: Biomedicine & pharmacotherapy = Biomedecine & pharmacotherapie

Article Title: Maduramicin-guided nanotherapy: A polymeric micelles for targeted drug delivery in canine mammary tumors.

doi: 10.1016/j.biopha.2023.116062

Figure Lengend Snippet: Fig. 7. Safety evaluation of MAD and MAD-TPGS in nude BALB/c mice bearing orthotopic tumors of CMT-U27 cells. Twenty nude BALB/c mice treated with saline, TPGS, MAD (1.75 mg/kg) or MAD-TPGS (1.75 mg/kg) for 18 days (n = 5) by oral administration or tail vein injection. (A) Body weight changes of mice were recorded throughout the whole period after oral administration or (D) tail vein injection (n = 5). (B) Representative images of primary organs in mice from H&E staining for oral administration or (E) tail vein injection. Scale bars = 100 µm. (C) Serum creatine kinase (CK), serum aspartate transaminase (AST), alanine transaminase (ALT) and blood urea nitrogen (BUN) levels of mice treated with oral administration or (F) tail vein injection (n = 3). * P < 0.05 for MAD vs other groups.

Article Snippet: MAD, TPGS, the physical mixture of MAD and TPGS, sucrose and lyophilized MAD-TPGS were measured via XRD (D8 Advance, Bruker-AXS, Karlsruhe, Germany) X.

Techniques: Saline, Injection, Staining